Method for determining the sex of birds

ABSTRACT

The present invention pertains to a method for determining nucleotides of a bird&#39;s W specific chromosome. The method comprises the steps of (a) obtaining a DNA sequence which includes the W specific chromosome from the bird. Then, there is the step of identifying nucleotides of the DNA sequence of the W specific chromosome. The present invention also pertains to a method of determining a sex of a bird, and a method of determining genetic relatedness between two birds. The present invention also pertains to a method for identifying a bird. Moreover, the present invention pertains to a method for determining predictive pairing for the genetic diversity of two birds. In another embodiment, the present invention pertains to a method for determining reproductive competence of a bird. In yet another embodiment, the present invention pertains to a method for predicting reproductive output of two or more birds. The present invention also pertains to a method for determining nucleotides of a bird&#39;s W specific chromosome. The method comprises the step of obtaining biological material of the bird. Then, there is the step of introducing a desired microsatellite probe to the biological material so that the W specific chromosome is indicated.

This is a continuation of application Ser. No. 07/999,229 filed on Dec.31, 1992, now abandoned.

FIELD OF THE INVENTION

The present invention is related to diagnosing birds. More specifically,the present invention is related to identifying aspects of the DNA of abird to identify it, its sex, its relationship with respect to otherbirds, and predictive of reproductive competence and reproductiveoutput.

BACKGROUND OF THE INVENTION

Many species of birds, being sexually monomorphic, do not displayphenotypic differences between the sexes especially in immatureindividuals. This is true for some commercial species (i.e., parrots andratites), and for certain endangered species, such as the CaliforniaCondor (Gynnogyps californianus), and Whooping Crane (Grus americana).Other non-endangered species, such as the Canada Goose (BrantaCanadensis), do not manifest sex-specific phenotypes.

Gender determinations can be performed in sexually monomorphic birdsthrough surgical examination. However, this procedure is less practicalwhen dealing with expensive or endangered species because it is invasiveand places the individuals being tested at risk. Alternatively, sexidentification can sometimes be made through karyotype analysis.However, karyotyping may be unreliable due to difficulties in obtainingavian chromosome spreads, lack of distinguishable sex chromosomes, orboth (Prus, S. E., and S. M. Schmutz. 1987. Comparative efficiency andaccuracy of surgical and cytogenetic sexing in psitticines. AvianDiseases 31:420-424).

The inability to identify gender in a reliable and non-invasive mannerin some birds represents an impediment to captive propagation programs,whether such programs are designed for commercial or restorationpurposes. Of value would be the development of DNA-based gender testsfor birds. Because the red blood cells of birds contain nuclei, only asmall volume of blood (10-100 ul) is required to provide sufficient DNAfor multiple tests (Longmire, J. L., A. K. Lewis, N. C. Brown, J. M.Buckingham, L. M. Clark, M. D. Jones, L. J. Meincke, J. M. Meyne, R. L.Ratliff, F. A. Ray, R. P. Wagner, and R. K. Moyzis. 1988. Isolation andmolecular characterization of a highly polymorphic centromeric tandemrepeat in the family Falconidae Genomics 2:14-24). Such volumes of bloodcan be drawn easily and safely by venipuncture, or collected from asimple nail-clipping procedure. In addition, many endangered arianspecies are already the subjects of DNA fingerprinting studies todetermine levels of genetic diversity and relatedness within natural andcaptive populations (Brock, M. K. and B. N. White. 1991. Multifragmentalleles in DNA fingerprints of the Parrot, Amazona ventralis. J. ofHeredity 82: 209-212; Longmire, J. L., G. F. Gee, C. L. Hardekopf, andG. A. Mark. Establishing paternity in whooping cranes (Grus americana)by DNA fingerprint analysis. The Auk, in press; Geyer, C. J., O. A.Ryder, L. G. Chennick, and E. A. Thompson. 1992. Analysis of relatednessin California condors from DNA fingerprints. Submitted to Mol. Biol.Evol; Maltbie, M. 1992. DNA fingerprints as a measure of geneticsimilarity in the endangered species, Attwater's Prairie-Chicken. M. S.Thesis, Texas Tech University). Thus, it would be helpful to identifyDNA sequence probes that enable sex identification in the same analysesthat generate highly polymorphic patterns achieved in DNAfingerprinting.

Microsatellites consisting of simple repeating nucleotide sequencesfound ubiquitously in the genomes of higher organisms might be used toachieve this goal. Simple repeats are commonly used to generate DNAfingerprints (Epplen, J. T., H. Ammer, C. Epplen, C. Kammerbauer, R.Mitreiter, L. Roewer, W. Schwaiger, V. Steimle, H. Zischler, E. Albert,A. Andreas, B. Beyermann, W. Meyer, J. Buitkamp, I. Nanda, M. Schmid, P.Nurnberg, S. D. J. Pena, H. Poche, W. Sprecher, M. Schartl, K. Weising,and A. Yassouridis. 1991. Oligonucleotide fingerprinting using simplerepeat motifs: A convenient, ubiquitously applicable method to detecthypervariability for multiple purposes. In; DNA fingerprinting:Approaches and applications (T. Burke, G. Dolf, A. Jeffreys, and R.Wolff, Eds). Birkhauser Press, Basil), and certain microsatellites havebeen previously shown to be sex-linked in some species. Kashi, Y., F.Iraqi, Y. Tikochinski, B. Ruzitski, A. Nave, J. S. Beckmann, A.Freidmann, M. Soller and Y. Gruenbaum. (1990). (TG)n uncoverssex-specific hybridization pattern in cattle. Genomics 7:31-36 reportedthat the dinucleotide repeat (TG)_(n) revealed sex-specific restrictionpatterns in cattle. The tetranucleotide repeat (GACA)₄ was found toprovide sex identification in certain reptiles (Epplen, J. T., H. Ammer,C. Epplen, C. Kammerbauer, R. Mitreiter, L. Roewer, W. Schwaiger, V.Steimle, H. Zischler, E. Albert, A. Andreas, B. Beyermann, W. Meyer, J.Buitkamp, I. Nanda, M. Schmid, P. Nurnberg, S. D. J. Pena, H. Poche, W.Sprecher, M. Schartl, K. Weising, and A. Yassouridis. 1991.Oligonucleotide fingerprinting using simple repeat motifs: A convenient,ubiquitously applicable method to detect hypervariability for multiplepurposes. In; DNA fingerprinting: Approaches and applications (T. Burke,G. Dolf, A. Jeffreys, and R. Wolff, Eds). Birkhauser Press, Basil). Inbirds, the M13 minisatellite detected female-specific restrictionpatterns in Mauritious Kestrels (Falco punctatus) and Peregrine Falcons(Falco peregrinus) (Longmire, J. L., R. E. Ambrose, N. C. Brown, T. J.Cade, T. L. Maechtle, W. S. Seegar, F. P. Ward and C. M. White. 1991.Use of sex-linked minisatellite fragments to investigate geneticdifferentiation and migration of North American populations of theperegrine falcon (Falcon peregrinus). Pages 217-229. In; DNAfingerprinting: Approaches and applications (T. Burke, G. Dolf, A.Jeffreys, and R. Wolff, Eds). Birkhauser Press, Basil), and thetrinucleotide repeat (CTT)_(n) has been shown to be sex-linked inpigeons and in chickens (Epplen, J. T., H. Ammer, C. Epplen, C.Kammerbauer, R. Mitreiter, L. Roewer, W. Schwaiger, V. Steimle, H.Zischler, E. Albert, A. Andreas, B. Beyermann, W. Meyer, J. Buitkamp, I.Nanda, M. Schmid, P. Nurnberg, S. D. J. Pena, H. Poche, W. Sprecher, M.Schartl, K. Weising, and A. Yassouridis. 1991. Oligonucleotidefingerprinting using simple repeat motifs: A convenient, ubiquitouslyapplicable method to detect hypervariability for multiple purposes. In;DNA fingerprinting: Approaches and applications (T. Burke, G. Dolf, A.Jeffreys, and R. Wolff, Eds). Birkhauser Press, Basil).

The present invention is directed to the use of sexing probes preferablybased on microsatellite repeats.

SUMMARY OF THE INVENTION

The present invention pertains to a method for determining nucleotidesof a bird's W specific chromosome. The method comprises the steps of (a)obtaining a DNA sequence which is present on the W specific chromosomefrom the bird. Then, there is the step of identifying nucleotides of theDNA sequence of the W specific chromosome.

The present invention also pertains to a method of determining a sex ofa bird, and a method of determining genetic relatedness between twobirds. The present invention also pertains to a method for identifying abird. Moreover, the present invention pertains to a method fordetermining predictive pairing for the genetic diversity of two birds.In another embodiment, the present invention pertains to a method fordetermining reproductive competence of a bird. In yet anotherembodiment, the present invention pertains to a method for predictingreproductive output of two or more birds associated with problems ofinbreeding depression.

The present invention also pertains to a method for determiningnucleotides of a bird's W specific chromosome. The method comprises thestep of obtaining biological material of the bird. Then, there is thestep of introducing a desired microsatellite probe to the biologicalmaterial so that the W specific chromosome is indicated.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings, the preferred embodiment of the inventionand preferred methods of practicing the invention are illustrated inwhich:

FIGS. 1a-1c are representative blots showing sex-specific DNAhybridization patterns. Of each DNA, 10 ug was digested with excessrestriction enzyme and electrophoresed within 0.8% agarose gels.Resulting southern blots were hybridized to microsatellite probes. FIG.1a is of Hinfl digested Canada Goose DNAs hybridized to (CT)_(n). FIG.1b is of Peregrine Falcon (P.F.) and California Condor (C.C.) DNAsdigested with HaeIII and hybridized to (CT)_(n). FIG. 1c is of HaeIIIdigested Peregrine Falcon DNAs hybridized to (GT)_(n). In each panel,males designated M, and females designated F. Size standards ( )included undigested, and HindIII digested bacteriophage lambda DNA, andHaeIII digested 0X174 DNA. Female-specific fragments indicated in eachpanel by dashes at approximately 50 kb.

FIG. 2 is a photograph of chromosomes of a female individual of theCalifornia condor.

DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention pertains to a method for determining nucleotidesof a bird's W specific chromosome. The method comprises the steps of (a)obtaining a DNA sequence which is present on the W specific chromosomefrom the bird. There is also the step of (b) identifying nucleotides ofthe DNA sequence of the W specific chromosome.

The step (b) of identifying nucleotides can include the steps of (c)separating fragments of the DNA sequence by size. Then, there is thestep (d) of tagging the fragments of the DNA sequence of interest. Next,there is the step (e) of displaying the tagged fragments so thediagnostic specific nucleotide sequences can be identified.

The step (c) of separating fragments can include the step (f) ofseparating electrophoretically fragments of the DNA sequence by size.The step (d) of tagging the fragments can include the step (g) ofhybridizing the fragments of DNA sequence. Furthermore, the step (e) ofdisplaying the tagged fragments can include the step (h) of taking apicture, preferably an x-ray picture of the DNA sequence such that thedesired fragments appear as bands on the x-ray picture. Preferably, thestep (g) of hybridizing the fragments includes the step (i) ofintroducing desired microsatellite probes to the fragments of the DNAsequence.

After the step (h) of taking an x-ray picture, there can be the step (j)of determining the bird is female if an intense band corresponding to amolecular weight of about 50 kilobases is present in the x-ray picture.

Alternatively, before the step (h) of taking an x-ray picture, there canbe the step (k) of repeating steps (a)-(g) and (i) for other birds. Inthis embodiment, the step (h) includes the step (l) of taking the x-raypicture so bands associated with all the birds are included in it.Preferably, after the step (l) of taking the x-ray picture, there is thestep (m) of scoring all readable bands and determining their frequencyamong a given bird taxon.

After the step (l) of taking the x-ray picture, there is the step (m) ofscoring all readable bands and determining an amount of bandsharing in apairwise comparison of the birds.

Moreover, the step (a) of obtaining a DNA sequence can include the step(m) of collecting biological material of the bird having DNA. The step(m) of collecting biological material can include the step (n) ofobtaining a cell from the biological material. Then, there can beincluded the step (o) of separating DNA from the cell. Additionally,there can be the step (p) of applying restriction enzyme to the DNA toseparate the DNA sequence from the DNA.

The present invention also pertains to a method for determining the sexof a bird. The method comprises the steps of obtaining biologicalmaterial of the bird. Then, there is the step of identifying the sex ofthe bird from the biological material. Preferably, the biologicalmaterial includes a desired DNA sequence which is present on the Wspecific chromosome. The step of obtaining biological material of thebird can be the obtaining step which is described above. The step ofidentifying the sex of the bird and the biological material can also bethe associated identifying step described above and specifically thestep (j) of determining if the bird is female.

Additionally, the present invention pertains to a method for determininggenetic relatedness between two birds. The method comprises the steps ofobtaining a DNA sequence from each bird. This step of obtaining a DNAsequence from each bird can preferably be the same as the obtaining stepdescribed above with respect to a single bird. Then, there is the stepof separating fragments of each DNA sequence by size. The step ofseparating fragments can preferably be the same as the separating stepdescribed above. Then, there is the step of comparing the fragments ofeach DNA sequence to identify whether they are substantially in common.This latter step uses the percentage of shared fragments. Closelyrelated birds (brothers, sisters, offspring) share a greater number ofbands than distantly related birds. For instance, if 40% or greater offragments are shared, the birds are substantially in common. Typically,the percentage of shared fragments is determined on a case by casebasis. First, for example, known brothers and sisters can be reviewedfor a general percentage of shared fragments. Then, known birds of thesubject bird taxon which are unrelated can be reviewed to determine apercentage of shared bands for unrelated birds relative to shared bandsof the related birds. From this information, a standard can beestablished which can be used as a basis for comparison for subsequentlyanalyzed unknown birds of that taxon.

Moreover, the present invention also pertains to a method foridentifying a bird. The method comprises the steps of obtaining a DNAsequence from the bird. Preferably, the obtaining step can be the sameas the obtaining step described above. Then, there is the step ofseparating fragments of the DNA sequence by size. Preferably, the stepof separating fragments can be the same as the separating step describedabove. Then, there is the step of hybridizing the fragments with desiredmicrosatellite probes. Next, there is the step of recording locations ofthe fragments. At some later time, the recorded locations can bereviewed with subsequently recorded locations obtained by the abovesteps to determine if the bird is the same bird as identified earlier.In samples from the same individual, all bands will be shared.

The present invention pertains to a method for determining predictivepairing for genetic diversity of two birds. The method comprises thesteps of obtaining a DNA sequence from each bird. The obtaining step canbe the same as the obtaining step described above. Then, there is thestep of separating fragments of each DNA sequence by size. Theseparating step can be the same separating step that is described above.Then, there is the step of hybridizing the fragments with desiredmicrosatellite probes. Next, there is the step of comparing thefragments of each DNA sequence to identify whether they aresubstantially dissimilar. For each taxon, the number of bands present isidentified and through determination of the frequency of each band inthe population, the relatedness of any two individuals can be predicted.Essentially, the determination of dissimilarity is the same as describedabove for commonality, but the focus is now on how unrelated are twobirds.

Moreover, the present invention pertains to a method for determiningreproductive competence of a bird. The method comprises the steps ofobtaining a DNA sequence which includes a W specific chromosome from thebird. The obtaining step can be the same obtaining step as describedabove. Then, there is the step of hybridizing the DNA sequence withdesired microsatellite probes to label, preferably the fragment from,the W specific chromosome. Next, there is the step of taking a pictureof the label associated with the W specific chromosome. The picturetaking step can be the same picture taking step as described above.Next, there is the step of measuring intensity of the label. Then, thereis the step of comparing intensity of the label against intensity oflabels of a W specific chromosome of reproductively competent males andfemales. For instance, a table of intensities can be created with knownsexed birds and used as a basis for comparison of a corresponding labelfor a given bird. For purposes herein, competence refers to either maleor female distinctiveness, or lack thereof (intersexed-sexually mixedbirds which can be expected to have limited reproductive capability).

The present invention pertains to a method for predicting reproductiveoutput of two or more birds. Reproductive output, for purposes herein,is understood to refer to the number of eggs a mating pair produces, thefertility of the eggs the mating pair produces, and the survivorship ofthe eggs and subsequent offspring of the mating pair. The methodcomprises the steps of obtaining a DNA sequence from each bird. Theobtaining step can be the same obtaining step as described above. Then,there is the step of separating fragments of each DNA sequence by size.The separating step can be the same separating step as described above.Next, there is the step of hybridizing the fragments with desiredmicrosatellite probes. Then, there is the step of comparing thefragments of each DNA sequence to known DNA fragments, preferably in atable, with respect to reproductive output to maximize production ofoffspring. For instance, mated pairs of birds which have been studiedover time with respect to the number of eggs, fertility and thesurvivorship thereof can be used to create a table of known DNAfragments with respect to the bird's reproductive output. This table offragments will then reflect the known reproductive output of mated pairsof birds.

In the operation of the preferred embodiment, the obtaining steppreferably includes the following steps.

1) Source of DNA

About 0.25-0.50 ml of freshly collected blood, macerated pieces oftissues (1/2 to 1/3 grams), or any other tissue that contains nucleatedcells can be used as a source of DNA. This includes blood left insideegg shells that remains from a newly hatched chick.

2) Lysis of Cell Membranes

The tissue is placed in 5 ml of lysis buffer (0.1M Tris-HCl pH 8.0, 0.1Methylenediamine-tetraacetic acid (EDTA), 0.01M NaCl, 0.5% (w/v) sodiumdodecyl sulfate (SDS)) contained in sterile 15 ml polypropylene tubesand shaken. This frees the DNA from inside the cell and permits it to bein solution in the buffer. Proteinase K (10 mg/ml) is added at this timeto the buffer if tissue other than blood is used. Samples can be storedin lysis buffer at room temperature for long periods of time.

3) Destruction of Proteins

To remove proteins from the solution containing the DNA, proteinase K(10 mg/ml) is added to 0.5 mg per ml, and the samples are incubated at37° C. on a tube rotator (slow rotation) overnight.

4) Extraction of DNA

An equal volume of phenol (molecular grade, preheated to ⁻ 50° C.)saturated in 1× TE buffer (100× TE make 484.4 grams of Tris, 152 gramsof EDTA and bring up to volume of 4 liters with distilled water) isadded to each sample (after overnight incubation with proteinase K). Thephenol/DNA buffer solution is placed on a rotator at room temperaturefor 30 minutes. This is followed by centrifugation for 5 minutes at 2000rpm.

The top layer (aqueous layer) is removed from the centrifuge tube andplaced in dialysis tubing (molecular weight cut off 12,000-14,000) andthe samples are then dialyzed in 1× TE at 4° C. against 3-4 changes of1× TE or longer until the smell of phenol can no longer be detected.

5) Cleaning Up of Samples

A) If samples did not appear clear at this step, they are put throughsteps 3-4 again until samples appear clean.

B) The undigested samples of DNA from an individual must be greater than50 kb in size if the sex identification is possible so this next step isto determine the molecular weight and the purity of DNA samples so nofalse male samples will be identified. Therefore, undigested samples ofDNA are electrophoresed on an agarose gel and compared to a 40 kb sizemarker.

The step of identifying nucleotides of the DNA sequence preferablyincludes the following steps.

6) Preparation of Gel for Electrophorese

An 8% agarose gel is prepared (350 ml of 1× TAE buffer (49.3 Trizmabase, 4.1 grams EDTA, 9.1 ml of Acetic acid, and fill to 1 liter pH to8.2 if needed) and 2.8 grams of agarose, boil for 3 minutes, allow tocool to about 60° with stirring, and then pour into 20 cm×25 cm gel boxwith an appropriate comb to produce 20 wells that will holdapproximately 42 μl of DNA solution and allow to set for one hour).

A 10 μl alloquot of each sample is added to 2 μl of 6× bluecrose andpipitted into a well.

Samples are loaded into the 8% agarose gel, usually with 2 to 3 markers(100 ng uncut lambda DNA and 500 ng Hind III cut lambda DNA) placed insome of the wells.

7) Electrophoreses of DNA Samples

The gel is run at 40 volts in 1× TAE buffer for about 3 hours. This isdone to visualize the DNA samples. DNA samples can be visualized with anultra-violet light source and photographed with polaroid film (Polaroidinstant film, type 57, 3000 speed). High molecular weight DNA (over 40to 50 kilobases) that appears clean of RNA is ready to go to the nextstep. If samples have a significant amount of RNA (which is common iftissues other than blood are used), they are put through a RNApurification step. RNA appears as small sized fragments on the gel.

8) RNA Purification Step

A 1/10 volume of RNase (1 mg/ml) is added to the sample and put in 37°C. incubator for 2 to 3 hours, then a half volume of 10% SDS is added tostop the reaction. Step 4 of phenol extraction, described above, isrepeated.

9) Preparation of DNA in Known Quantities (300 μg/ml)

Clean, high molecular weight DNA Is quantified by UV spectroscopy(Maniatus et al., 1982, incorporated by reference). Dilution of samplesfor spectroscopy is 25 μl of sample plus 475 μl of 1× TE. The A²⁶⁰reading is multiplied by 1000 to give the concentration of the DNAsamples in μg/ml. Samples are adjusted to 300 μg/ml by precipitating outthe DNA by adding 0.1 sample volume of 3M sodium acetate and 2.5-3 timessample volume cold (0° C.) 200 proof ethanol. The concentration of theDNA samples multiplied by its original volume after dialysis is thendivided by 300 to indicate the amount of 1× TE that is needed to beadded to get final concentration of 300 μg/ml. After 1× TE is added,samples are put on rotator overnight at 4° C. and then stored at 4° C.when not in use.

10) Digestions of DNA with Restriction Enzymes

A digestion is set up for each sample: 33 μl or 10 micrograms of DNAsample, 4 μl of 10× restriction enzyme buffer (Promega buffer B), and 3μl of restriction enzyme (Promega HinfI). Digestions are well mixed andtough agitated before being placed in 37° C. incubator overnight.

Enzyme digestion is stopped through heat killing. Digestions are placedin 70° C. for 10 minutes.

11) Gel Electrophoreses for Southern Analysis

An 8% agarose gel is prepared (350 ml of 1× TAE and 2.8 grams ofagarose, boil for 3 minutes, allow to cool to ⁻ 60° with stirring, pourinto 20 cm×25 cm gel box and allow to set for 1 hour), then submerge in1× TAE buffer in gel rig.

Four microliters of 6× bluecross is mixed with each digested sample.

Two to three wells are filled with 20 μl of mixed marker plus 2 μl of 6×bluecross.

Gels usually have 20 wells, 17 wells for samples and 3 wells formarkers. Markers are set up so they are asymmetrical. The location ofsamples can then be easily assessed and the order of samples has minimalprobability of being confused.

A 41 μl of sample of a given bird is loaded into a well. A total of 17birds can be run (loaded) on a single gel with 3 wells reserved formolecular weight markers. A 21 μl sample of marker is loaded into eachof the 3 marker wells.

The gel is run at 40 volts for about 36 to 48 hours.

After completion of a run, a polaroid photograph using ultraviolet lightis taken with a red filter to document the extent of digestion and thatmovement through gel is normal.

12) Southern Transfer

A) Gels are submerged for 5 minutes in 0.25M HCl, followed by two15-minute submersions in 0.4M NaOH (fresh NaOH each submersion). A stirbar is placed in the middle of the gel box for each submersion to keepliquid moving.

B) Restriction fragments are transferred to a nylon membrane (BoehringerMannheim) using the procedure of Southern (1975), incorporated byreference.

C) Transfers are allowed to take place overnight and then taken apart.Membranes are washed gently by hand in 2× SSC, followed by one 15 minutewash in a neutralization solution (0.5M Tris pH 7.5, 1.5M NaCl). This isfollowed by two 15-minute washes in 2×SSC. All these washes are done atroom temperature on an orbital mixer. Membranes are dried between twoclean sheets of Whatman paper (3 MM chromatography paper) and stored inplastic covering until used.

13) Hybridization

Preparation of the Southern Transfer Membrane

A) Immediately prior to hybridization, membranes are washed in 0.1×SSC,0.1% SDS at 60° C. for 1 hour on an orbital mixer.

B) Membranes are then treated with a prehybridization solution at 42° C.for 45 minutes to 1 hour on an orbital mixer. Prehybridization mixturefor 1 to 2 membranes in a sealed bag is made of a total of a 100 ml ofthe following ingredients: 30 ml 20× SSC, 35 ml formamide, 1 ml 0.5MEDTA, 5 ml 20% SDS, 0.25 grams of powdered milk, and brought up tovolume of 100 ml with distilled water. For more than 2 membranes, theyare put in a tub (usually Tubberware) with enough prehybridizationsolution so that membranes are submerged. Prehybridation solution isstirred and warmed until clear before it was put on membranes.

Nick Translation of the DNA Probe

DNA probes are labeled with ³² P as follows: 1.0 μg probe DNA (in thisprocedure it is poly (dA-dG)·(dC-dT) supplied by Pharmacia), 5 μl ofmarker, 1 μl each of cold dATP, dGTP, and dTTp, 5 μl ³² P dCTP, 5 μl of10× nick translation buffer (0.5M TrisCl (pH 7.5), 0.1M MgSO₄, 1 mMdithiothreitol, 5 μg/ml bovine serum albumin), 5 μl DNase/Polymerase I,and distilled water is added to make 50 μl total). This solution isincubated for 45 minutes at 15° C. Solutions of 50 μl 1× TE and 10 μl0.5% SDS are then added to the probe and then run through a G-50 in 1×TE sephadex spin column by centrifugation. After this, the labeled probeis treated with 1/9 volume of 1M NaOH and then incubates for 10 minutesat 37° C. The probe is added directly to the prehybridization solutioncontaining the membranes and allowed to hybridize overnight at 42° C. onan orbital mixer. A kit for labeling a probe is sold by ClontechLaboratories, Inc. of Palo Alto, Calif. which has full instructionstherein to accomplish the same, incorporated by reference. (Note, a BTbuffer is comprised of 8.4 grams of sodium bicarbonate, 17.53 grams ofsodium chloride and 0.5 ml Tween 20 (Sigma Co.). Alternatively, thetrinucleotide repeat (CTT)_(n) was labeled with ³² P!dCTP using theprimer extension method described by Feinberg, A. P., and B. Vogelstein.1983. A technique for radiolabeling DNA restriction fragments to highspecific activities. Anal. Biochem. 132:6-13), incorporated byreference.

Following overnight hybridization, membranes are rinsed in 2× SSC atroom temperature followed by a 15-minute wash in 2× SSC on an orbitalmixer at room temperature. Membranes are then washed twice for 15minutes each in 1× SSC, 0.1% SDS at 50° C. on an orbital mixer.Membranes are blotted dry with a clean sheets of Whatman paper andwrapped in saran wrap without wrinkles.

Exposure of Film with the Radioactive Probe

The membranes are then placed in a cassette with intensifying screensand a piece of film (Kodak XAR-5) and exposed overnight at -70° C. Ifafter overnight exposure, the film is inadequately exposed a secondpiece of film is exposed for a longer time frame. The length of exposureis varied until the desired level of exposure is obtained.

Scoring of Autoradiograms (See DNA fingerprints as a measure of geneticsimilarity in the endangered species, Attwater's Prairie-Chicken. M. S.Thesis, Texas Tech University, incorporated by reference)

There are two ways to score autoradiograms. One method is to just scorepolymorphic fragments and to calculate their frequency among a birdtaxon. The polymorphic fragment frequency is calculated as the totalnumber of individuals with a specific band (not shared among allindividuals) divided by the total number of individuals. This frequencyis the average of all polymorphic bands scored on the autoradiogram andcould then be compared to other frequencies for different species ofbirds in work done by other investigators using DNA fingerprinting(Burke and Bruford, 1987; Longmire et al., 1992, incorporated byreference). The second approach is to score all readable bands even ifthey are shared among all individuals. This allows calculation of anindex of similarity among all individuals in a pairwise comparison usingthe same DNA fingerprinting probe and digested with the same enzyme. Theformula used to calculate the amount of band sharing in a pairwisecomparison is the total number of bands shared by two birds in thecomparison divided by their total number of bands (Wetton et al., 1987,incorporated by reference). This will allow the investigator to seewhich bird is more varied from the other birds in the comparison usingthat specific enzyme/probe combination. To keep consistency in scoring,standard markers are run at both ends and the middle of the gel, andsometimes samples from individual birds are duplicated at opposite endsof the gel so that the chance of error could be reduced.

DNA Marker

For many birds, the microsatellite probe, (TC)_(n), is the probe that isable to identify the sex marker of an individual. The sex marker is avery intense band of high molecular weight estimated to be about 50kilobases in female birds.

Results include the observation of high molecular weight,female-specific restriction fragments at approximately 50 kilobase pairs(kb) in the majority of species that were tested following digestionwith certain restriction enzymes and hybridization to variousmicrosatellite probes (Table 1, FIG. 1). The 50 kb fragment has beenshown to be present on the W specific chromosome in the Californiacondor (see FIG. 2) and its presence on the W specific chromosome is thebasis for gender identification.

                  TABLE 1                                                         ______________________________________                                        Sex identification in birds using various microsatellite                      probes. Numbers indicate restriction enzymes successfully used to             reveal high molecular weight, female-specific, microsatellite                 fragments (1 = HaeIII; 2 = Hinfl; 3 = Pstl). Zero (0) indicates               female-specific microsatellite fragments not detected. Asterisks              indicate reduced hybridization signal intensity. ND-not done.                             Microsatellite Probe                                              Species       (CT)n   (GT)n     (CG)n (CTT)n                                  ______________________________________                                        Peregrine Falcon                                                                            1       1         0*    0*                                      (9 F, 16 M)                                                                   California Condor                                                                           1,2     0         0*    0                                       (6 F, 3 M)                                                                    Canada Goose  2       0         0*    2                                       (14 F, 5 M)                                                                   House Sparrow 0       0         0*    0                                       (5 F, 5 M)                                                                    Wild Turkey   1,3     0         0*    ND                                      (7 F, 4 M)                                                                    Attwater Prairie-Chicken                                                                    1       0         0*    1                                       (2 F, 14 M)                                                                   Greater Prairie-Chicken                                                                     1,2     0         0*    1,2                                     (9 F, 6 M)                                                                    Chattering Lory                                                                             0       ND        0*    0                                       (2 F, 2 M)                                                                    Whooping Crane                                                                              0       0         0*    0*                                      (5 F, 5 M)                                                                    ______________________________________                                    

Dinucleotide repeat (CT)_(n) revealed sex-specific fragments in HaeIIIdigested Peregrine Falcon DNA; California Condor DNA digested withHaeIII or Hinfl; Canada Goose DNA digested with Hinfl; Wild Turkey(Meleagris gallopazo) DNA digested with HaeIII or Pstl; Attwater'sPrairie-Chicken (Tympanuchus cupido attwateri) DNA digested with Hinflor HaeIII. Dinucleotide repeat (GT)_(n) revealed female-specific HaeIIIrestriction fragments in the Peregrine. Trinucleotide repeat (CTT)_(n)hybridized to female-specific Hinfl restriction fragments in the CanadaGoose and Attwater's Prairie Chicken DNA after digestion with HaeIII,and in Greater Prairie-Chicken DNA following digestion with HaeIII orHinfl. In the above cases, sex determination by DNA analysis matchedexactly to morphometric examination. All of the microsatellite probeshybridized to DNAs sufficiently to allow overnight autoradiographicexposures, except (CTT)_(n) which showed reduced hybridization to DNAfrom the Peregrine and Whooping Crane, and (CG)_(n) which producedlittle or no detectable hybridization to any DNA sample. In addition,each of the microsatellite probes, except (CG)_(n), revealed highlypolymorphic (DNA fingerprint) patterns in all species that were tested.

Of interest were the variable results obtained depending upon whichmicrosatellite probes were used with the different species. Among thebirds that displayed female-specific patterns in response to at leastone repeat probe, certain probes revealed sex-specific patterns in somespecies but not in others. For example, (CTT)_(n) revealed sex-specificpatterns in the Canada Goose and Prairie-Chickens, but not in thePeregrine or California Condor. On the other hand, only the Peregrinedisplayed sex-specific patterns following hybridization to (GT)_(n).Only (CG)_(n) was non-informative in all species. The observation that(CG)_(n) did not hybridize to these avian DNAs is not surprisingconsidering that CG is the rarest dinucleotide within vertebrategenomes, and probably does not exist as a moderate copy-number repeat(Stallings, R. L. 1992. CpG suppression in vertebrate genomes does notaccount for the rarity of (CpG)n microsatellite repeats. Genomics17:890-891; Sved, J., and A, Bird. 1990. The expected equilibrium of theCpG dinucleotide in vertebrate genomes under a mutational model. Proc.Natl. Acad. Sci. USA 87:4692-4696).

The set of microsatellite probes tested allowed unambiguous gendertesting in the Peregrine Falcon, California Condor, Wild Turkey, CanadaGoose, Greater Prairie-Chicken, and Attwater's Prairie-Chicken. Takinginto account that there are only 4 nucleotides present in DNA, circularpermutation of repeat sequences, as well as strand complementarity,there are 6 dinucleotide, 12 trinucleotide, 39 tetranucleotide, 109pentanucleotide, and 366 possible hexanucleotide repeat probes. Theseprobes can be produced using an Oligo nucleotide synthesizing machine orobtained from any of a variety of professional companies which providecustomized synthesis of DNA.

The procedure for discovering microsatellite probes that will work, for,as yet, untested species would consist of the following steps: by takinginto account the circular permutations of repeat sequences, as well asstrand complementarity of DNA, there are 6 dinucleotides, 12trinucleotides, 39 tetranucleotides, 109 pentanucleotides, and 366possible hexanucleotide repeat probes. There is a high probability thatall permutations will not be required as certain combinations are rarelyfound in the genome as tandem repeats; for example, GT and CT are morecommon in the genome than are GC and AT. After the relative frequency ofmicrosatellites is assessed for a sample of bird species (e.g., Table 1in Longmire et al., ms), then a bank of probes can be developed tomaximize the probability of gender identification in a more universalmanner and thereby reduce experimental procedures to identify theputative species-specific probe. The larger the data base, the less timewill be required to discover the correct probe for previouslyuninvestigated species.

In the instance of species in which one of the four dinucleotides failedto resolve gender of individuals, the nature (sequence) of themicrosatellite that resolves gender can be identified by digestion ofnuclear DNA samples with a combination of 4-base-cutter restrictionenzymes. The large molecular sized product on an agarose gel can beisolated and cloned and sequenced to identify the microsatellite(s)associated with the sex chromosomes.

The present invention also pertains to a method for determiningnucleotides of a bird's W specific chromosome. The method comprises thestep of obtaining biological material of the bird. Then, there is thestep of introducing a desired microsatellite probe to the biologicalmaterial so that the W specific chromosome is indicated. Preferably, thebiological material includes nucleated blood and the introducing stepincludes the step of hybridizing in situ the blood with the desiredmicrosatellite probe. The in situ hybridizing step is described inMoyzis, R. K., L. Albright, M. F. Bartholdi, L. S. Cram, L. L. Deaven,C. E. Hildebrand, N. E. Josta, J. L. Longmire, J. Meyne, and T. S.Robinson. 1987. Human chromosome-specific repetitive DNA sequences:Novel markers for genetic analysis. Chromosoma 95:375-386; Moyzis, R.K., J. M. Buckingham, L. S. Cram, L. L. Deaven, M. D. Jones, J. Meyne,R. L. Ratliff, and J. R. Wu. 1988. A highly conserved repetitive DNAsequence (TTAGGG)_(n), present at the telomeres of human chromosomes.Proc. Nat. Acad. Sci. USA 85:6622-6626, incorporated by reference.

FIG. 2 is a photograph of chromosomes of a female individual of theCalifornia condor showing the presence of the W specific chromosomeidentified by two yellow dots. The gross morphology of the W specificchromosome has been identified by karyotyping by Dr. Oliver Ryder of theSan Diego Zoo and the chromosome identified by yellow matches thedescription of the W specific chromosome described by Ryder. In males ofthe California condor, no such element is present. Note also that in theinterphase nucleus at the corner of the photo, the yellow indicating thepresence of the W specific chromosome can be seen. This means that inthis embodiment, biological material, and preferably interphase bloodcells, can be used to sex condors without any DNA isolation. Once theproper microsatellite has been identified for the W specific chromosomeof any bird taxon, this method can be applied to identifying the genderof individual birds of that taxon.

A system for any of the above methods (identification, sex, relationshipwith other birds, reproductive capability, etc.) can include the desiredmicrosatellite probes, a container for biological material, materialsnecessary to prepare the biological material for tagging with theprobes, and a mechanism to identify the presence of the probes, all ofwhich are consistent with the described methods above.

Although the invention has been described in detail in the foregoingembodiments for the purpose of illustration, it is to be understood thatsuch detail is solely for that purpose and that variations can be madetherein by those skilled in the art without departing from the spiritand scope of the invention except as it may be described by thefollowing claims.

What is claimed is:
 1. A method for determining the size of a specificnucleotide segment of either a Peregrine Falcon's, California Condor's,Canadian Goose's, Wild Turkey's, Attwater Prairie-Chicken's or a GreaterPrairie-Chicken's W specific chromosome comprising the steps of:(a)obtaining a DNA sequence which is present on the W specific chromosomefrom the bird; (aa) introducing microsatellite probes to the DNAsequence so they interact, said microsatellite probes being eitherdinucleotide, trinucleotide, tetranucleotide, pentanucleotide orhexanucleotide repeat probes; and (b) identifying nucleotides of the DNAsequence of the W specific chromosome from the probes which haveinteracted with the DNA sequence so nucleotides of the bird's W specificchromosome are determined.
 2. A method as described in claim 1 whereinstep (b) includes the steps of (c) separating fragments of the DNAsequence by size, (d) tagging the fragments of the DNA sequence ofinterest, and (e) displaying the tagged fragments so nucleotides can beidentified.
 3. A method as described in claim 2 wherein step (c)includes the step (f) of separating electrophoretically fragments of theDNA sequence by size; the step (d) includes the step (g) of hybridizingthe fragments of DNA sequence, and the step (e) includes the step (h) oftaking an x-ray picture of the DNA sequence such that the fragmentsappear as bands on the x-ray picture.
 4. A method as described in claim3 wherein step (g) includes the step (i) of introducing microsatelliteprobes to the fragments of the DNA sequence.
 5. A method as described inclaim 4 including after step (h), the step (i) of determining the birdis female if a band corresponding to a molecular weight of about 50kilobases is present the in x-ray picture.
 6. A method as described inclaim 4 including before the step (h), there is the step (k) ofrepeating steps (a)-(g) and (i) for other birds, wherein the step (h)includes the step (l) of taking the x-ray picture so bands associatedwith all the birds are included in it.
 7. A method as described in claim6 including after the step (l), there is the step (m) of scoring allreadable bands and determining their frequency among a given bird taxon.8. A method as described in claim 6 including after the step (l), thereis the step (n) of scoring all readable bands and determining an amountof band sharing in a pairwise comparison of individual birds.
 9. Amethod as described in claim 8 wherein step (a) includes the step (m) ofcollecting biological material of the bird having DNA.
 10. A method asdescribed in claim 9 wherein the step (m) includes the step (n) ofobtaining a cell from the biological material, the step (o) ofseparating DNA from the cell, and the step (p) of applying restrictionenzyme to the DNA to separate the DNA sequence from the DNA.
 11. Amethod for determining genetic relatedness between two birds of either aPeregrine Falcon, California Condor, Canadian Goose, Wild Turkey,Attwater Prairie-Chicken or a Greater Prairie-Chicken comprising thesteps of:obtaining a DNA sequence which is present on a W specificchromosome from each bird; separating fragments of each DNA sequence bysize; hybridizing the fragments with microsatellite probes, saidmicrosatellite probes being either dinucleotide, trinucleotide,tetranucleotide, pentanucleotide or hexanucleotide repeat probes; andcomparing the fragments of each DNA sequence to identify whether theyare substantially in common so the genetic relatedness between the twobirds is determined.
 12. A method for identifying either a PeregrineFalcon, California Condor, Canadian Goose, Wild Turkey, AttwaterPrairie-Chicken or a Greater Prairie-Chicken comprising the stepsof:obtaining a DNA sequence which is present on a W specific chromosomefrom the bird; separating fragments of the DNA sequence by size;hybridizing the fragments with microsatellite probes, saidmicrosatellite probes being either dinucleotide, trinucleotide,tetranucleotide, pentanucleotide or hexanucleotide repeat probes; andrecording locations of the fragments so the bird is identified.
 13. Amethod for determining predictive pairing for genetic diversity of twobirds Of either a Peregrine Falcon, California Condor, Canadian Goose,Wild Turkey. Attwater Prairie-Chicken or a Greater Prairie-Chickencomprising the steps of:obtaining a DNA sequence which is present on a Wspecific chromosome from each bird; separating fragments of each DNAsequence by size; hybridizing the fragments with desired microsatelliteprobes, said microsatellite probes being either dinucleotide,trinucleotide, tetranucleotide, pentanucleotide or hexanucleotide repeatprobes; and comparing the fragments of each DNA sequence to identifywhether they are substantially dissimilar so predictive pairing forgenetic diversity is determined.
 14. A method for determiningreproductive competence of a bird comprising the steps of:obtaining aDNA sequence which includes a W specific chromosome from the bird;hybridizing the DNA sequence with microsatellite probes to label the Wspecific chromosome; taking a picture of the label associated with the Wspecific chromosome; measuring intensity of the label; and comparingintensity of the label against intensity of labels of W specificchromosome of reproductively competent males and females.
 15. A methodfor determining reproductive output of two or more birds of either aPeregrine Falcon, California Condor, Canadian Goose, Wild Turkey,Attwater Prairie-Chicken or a Greater Prairie-Chicken comprising thesteps of:obtaining a DNA sequence which is present on a W specificchromosome from each bird; separating fragments of each DNA sequence bysize; hybridizing the fragments with microsatellite probes, saidmicrosatellite probes being either dinucleotide, trinucleotide,tetranucleotide, pentanucleotide or hexanucleotide repeat probes; andcomparing the fragments of each DNA sequence to known DNA fragments withrespect to reproductive output to predict production of offspring soreproductive output of the two birds is determined.
 16. A method fordetermining the size of a specific nucleotide segment of either aPeregrine Falcon's, California Condor's, Canadian Goose's, WildTurkey's, Attwater Prairie-Chicken's or a Greater Prairie-Chicken's Wspecific chromosome comprising the steps of:obtaining nucleated blood ofthe bird; and hybridizing in situ the blood with a microsatellite probeso nucleotides of the bird's W specific chromosome are determined.
 17. Amethod as described in claim 1 including after step (aa), there is thestep of (g) digesting the DNA sequence with a combination of4-base-cutter restriction enzymes if the microsatellite probes do notreveal the gender of the bird.
 18. A method for determining the sex ofeither a Peregrine Falcon, California Condor, Canadian Goose, WildTurkey, Attwater Prairie-Chicken or a Greater Prairie-Chicken from therespective bird's w-specific chromosome comprising the stepsof:obtaining a DNA sequence which is present on the W specificchromosome from the bird; introducing microsatellite probes to the DNAsequence so they interact to reveal the sex of the bird, saidmicrosatellite probes being either dinucleotide, trinucleotide,tetranucleotide, pentanucleotide or hexanucleotide repeat probes; andidentifying the sex of the bird from the probes which have interactedwith the DNA sequence.
 19. A method as described in claim 18 includingafter the introducing step, there is the step of digesting the DNAsequence with a combination of 4-base-cutter restriction enzymes if themicrosatellite probes do not reveal the sex of the bird.
 20. A methodfor determining the sex of either a Peregrine Falcon, California Condor,Canadian Goose, Wild Turkey, Attwater Prairie-Chicken or a GreaterPrairie-Chicken comprising the steps of:obtaining nucleated blood of thebird; hybridizing in situ the blood with a microsatellite probe; andidentifying the sex of the bird from the hybridized blood.
 21. A methodas described in claim 10 wherein the probe is either (CT)n, (GT)n, (CG)nor (CTT)n.
 22. A method for determining which microsatellite probe willreveal the sex of either a Peregrine Falcon, California Condor, CanadianGoose, Wild Turkey, Attwater Prairie-Chicken or a GreaterPrairie-Chicken from the respective bird's W chromosome comprising thesteps of:introducing dinucleotide, trinucleotide, tetranucleotide,pentanucleotide and hexanucleotide repeat microsatellite probes to a DNAsequence having a W specific chromosome of the bird; assessing therelative frequency of each microsatellite probe that has labeled the DNAsequence; and forming a database identifying probability that a givenmicrosatellite will reveal a bird's sex, said probability correspondingto the relative frequency of the given microsatellite labeling the DNAsequence, so the database will identify which microsatellites can beused for a bird to determine the bird's sex.